Many biological and chemical properties of nano- and micro-sized objects, such as particles, electrodes, implants, bubbles, droplets, viruses, micelles, liposomes and living cells are determined by processes occurring at the interface between the object and a medium surrounding or being in contact with the object. Examples of interface processes include the electrostatic current that flows through the outer membrane of a neural cell, the transport of proteins and reagents through a membrane, production processes inside organelles such as ribosomes and mitochondria, the uptake of oxygen in blood cells, or the formation of fibrous plagues responsible or related to Alzheimer's disease. These interface processes are usually confined to a 1 nm thick slab or layer that surrounds the object. Linear optical techniques are insensitive to interface processes and cannot be used for a determination of the properties of the interface processes of these objects.
Second-order nonlinear optical techniques are surface sensitive and ideally suited to image interface processes in isotropic media. Second harmonic generation (SHG) is a second-order nonlinear optical process, in which two photons (usually with identical frequencies and originating from the same laser beam) interacting via a nonlinear process with a material are effectively combined to form new photons with twice the frequency and half the wavelength of the initial photons. This process is a special case of sum frequency generation and unlike fluorescence the process is instantaneous. For isotropic materials the SHG process is forbidden in bulk media but allowed at interfaces. Apart from being sensitive to the structure of an interface, there is also a linear relationship between the emitted electric field and the electromagnetic potential. This includes the sensitivity for surface plasmons. Accordingly, probing a bulk liquid such as water with regard to a SHG process only gives rise to a very weak background signal arising from small fluctuations in the average isotropy of the structure. The generation of the background signal is commonly known as Hyper Rayleigh Scattering (HRS).
An overview of applications and experiments using a SHG scattering signal is given in [1]. In many previous experiments [1-7] the measurement setup included a femtosecond laser source emitting a single beam of laser pulses with a high repetition rate in a range from 80-90 MHz with a wavelength of 800 nm and pulse energies of up to 15 nJ. The SHG scattered signal was detected with a photomultiplier tube in the photon counting mode. In many cases chromophores were included in the samples to enhance the signal strength of the SHG scattered signal and improve the detection efficiency. Chromophores stimulate the emission of the SHG scattered radiation if the SHG photon energy matches one of the energy levels of the chromophore.
In a few studies [6, 8] no emission enhancing chromophores have been utilized. The intrinsic signal response of the interfaces was directly measured, but the detected SHG signal was very low. A corresponding experiment published in [6] reports about the detection of SHG scattered radiation from the surface of a dilute suspension of 100 nm polystyrene particles in water.
A global description of SHG microscopy is given in [9]. Most SHG microscopy applications rely on the detection of exogenous markers such as surface modifiable SHG active nanoparticles or endogenous bulk responses [10]. One example of detecting surface properties with SHG microscopy is the measurement of the membrane potential in dendritic spines using a SHG signal [11]. In this example chromophores were engineered to exhibit a membrane affinity so that they could be used to enhance the SHG signal from the membrane. In a number of cases endogenous structures have been investigated that exhibit a non-centrosymmetric structure and therefore give rise to allowed bulk SHG [12]. The instrumentation used for SHG microscopy is similar to that used for SHG scattering measurements and the more commonly employed confocal two-photon fluorescence microscopy. The experimental optical setup in most experiments typically includes an oscillator as a light source for excitation with a pulse repetition rate of more than 1 MHz and the pulse energies of the laser pulses are typically below 10 nJ. The excitation light is focused tightly onto a spot in the sample. The spot, or the sample, is then scanned and the SHG scattered signal is detected using a photon counting technique [9], so that the position of the spot is associated to the detected signal to then produce two- or three-dimensional image. Examples of a label-free surface sensitive SHG microscopy have been described as well. It was the object of these experiments to detect the surface chiral response of a patterned planar supported lipid bi-layer [13]. Wide-field SHG microscopes have also been devised [14][15] which eliminate the need for scanning. A common approach is to use a light source for excitation that also consists of an oscillator with a 75 MHz repetition rate and an intensified CCD camera [14], or a very low repetition rate (1 kHz, using a chirped pulse amplifier) and a regular CCD camera providing a poor photon throughput [15].
In second harmonic and multi-photon detection and imaging, varying the phase, polarization and temporal component of the excitation provides additional information regarding the sample under investigation. In scanning systems the phase is typically varied with a spatial light modulator (SLM) [16], while the polarization is changed with a wave plate, so that only one dimension of the nonlinear tensor representing the nonlinear properties of the sample can be addressed at a time, and it is not possible to modify the temporal component of the excitation. In certain wide-field configurations [15], in which the excitation beam is split into two beams, it is possible to address two dimensions of the nonlinear tensor and the temporal component.
U.S. Pat. No. 6,055,051 A refers to a method for determining surface properties of microparticles. Second harmonic generation (SHG), sum frequency generation (SFG) and difference frequency generation (DFG) can be used for surface analysis or characterization of microparticles having a non-metallic surface feature. The microparticles can be centrosymmetric or such that non-metallic molecules of interest are centrosymmetrically distributed inside and outside the microparticles but not at the surface of the microparticles where the asymmetry aligns the molecules. The signal is quadratic in incident laser intensity or proportional to the product of two incident laser intensities for SFG, it is sharply peaked at the second harmonic wavelength, quadratic in the density of molecules adsorbed onto the microparticle surface, and linear in microparticles density.
WO 02 46764 A1 refers to methods of detecting molecules at an interface which comprise la-belling the molecules with a second harmonic-active moiety and detecting the labelled molecules at the interface using a surface selective technique. The disclosure also provides methods for detecting a molecule in a medium and for determining the orientation of a molecular species within a planar surface using a second harmonic-active moiety and a surface selective technique.
US 2010 031748 A1 discloses methods for detecting and evaluating the quality of protein crystals comprising subjecting a sample to second order non-linear optical imaging and detecting the second harmonic generation signal.
EP 07 40156 A1 relates to the use of nonlinear optical methods of surface second-harmonic generation and sum-frequency generation to detect immuno and enzyme reactions and nucleotide hybridisation.
In the majority of applications in which SHG signals have been used including SHG scattering experiments and SHG microscopic measurements chromophores such as fluorescent dyes, intrinsic or genetically modified proteins, quantum dots, and nanoparticles are utilized as beacons to enhance the strength of the SHG scattered light [1, 9].
However, the use of these photosensitive markers results in a low photo damage threshold of the sample material [15]. This in turn requires the use of low pulse energies of the exciting laser beam in order to avoid damages of the sample. In combination with low pulse energies high repetition rates of the laser pulses and a narrow laser focus are used in order to obtain sufficient signal strength that can be detected. Accordingly, to perform these measurements, pulse energies in the order of 0.1-1 nJ and repetition rates in the range MHz to GHz are typical for the laser sources [9]. For microscopy, these conditions often necessitate a confocal microscopy layout employing a narrow focus that is scanned across the sample. The need to scan the optical beam significantly limits the time resolution for imaging.
Most efforts to enhance the sensitivity of detection of SHG scattered signals either followed a direction to design and apply more efficient chromophores being less prone to photo damage or having a higher quantum efficiency, or a direction to optimize the pulse energy and repetition rate of the laser pulses with regard to the photosensitive markers. The latter optimization strategy resulted in scanning systems with (1) higher repetition rates (if the chromophores were already saturated by the applied laser pulse energies) or in (2) lower repetition rates to reduce thermal effects [9][19][20][21]. A study has been performed on the role that polarization, numerical aperture (NA), and wavelength has on the generation of second harmonic signal in collagen fibers [22]. In the latter study, it was found that lowering the NA, i.e. increasing the size of the illumination spot, decreases the signal. Other efforts relied on using a wide-field geometry with an accustomed high repetition rate (˜80 MHz) [14], or with a low repetition rate (<10 kHz) yielding a poor photon throughput [15]. Therefore, none of the previously mentioned approaches have resulted in a significantly increased photon throughput and have a reduced photon damage risk.